Studies have suggested that the presence of epithelial cells in the hematopoietic system indicates the spread of cancer from a localized area to other parts of the body (also known as metastisis). This discovery is important since metastisis is diagnostic of certain stages of cancer, and decisions concerning the proper treatment of a cancer patient are largely dependent upon properly characterizing the stage of the disease. In particular, treatments for patients having localized cancer can be vastly different from treatments given to patients in metastatic stages of cancer.
With the advent of nucleic acid amplification reactions such as the polymerase chain reaction (PCR), epithelial cells present in the hematopoietic system can be detected at the nucleic acid level instead of at the protein level. Hence, problems associated with crossreactive antibodies are avoided. Additionally, it is well known that nucleic acid amplification reactions are significantly more sensitive than more conventional antibody based assay methods. Amplification based assays for detecting epithelial cells in the blood stream have therefore provided significant advantages over immunocytological assay methods for detecting early stages of metastatic cancer.
PCR based assays employed to detect epithelial cells in the hematopoletic system have been reported in the literature. Most of these assays target a nucleic acid sequence encoding cytokeratin 19 (CK19), a protein found on the surface of epithelial cells. However, many psuedogenes (comprising a nucleic acid sequence that closely mimics the gene for CK19) are present in the human genome. Thus, one challenge facing those developing amplification assays to detect a CK19 target sequence is to design assays that amplify and detect a sequence from the CK19 gene but not the closely related pseudogene.
Additionally, it is well known that amplification primer sequences can be selected based upon computer comparisons of closely related sequences. Theoretically, sequences selected in this manner effectively should produce copies of the selected target sequence when employed according to nucleic acid amplification principles. Notwithstanding, the theoretical efficacy of sequences selected in the above manner, it is often times true that such sequences do not produce acceptable amounts of amplification product. Unfortunately, this phenomenon is not understood. Accordingly, while primers initially can be screened using computer programs efficacy cannot be adequately determined until such primers are employed in practice.
A further challenge faces those designing PCR assays that use microparticle capture based detection procedures for detecting amplification products. Specifically, amplified target sequences detected with the assistance of microparticles must be sufficiently short so that amplification product captured on the microparticle does not interfere with the capture of additional amplification product. Accordingly, those choosing to detect amplification products with the assistance of a microparticle are faced with an added restriction in terms of selection of a suitable target sequence. In particular, suitable target sequences are constrained to sequences that are relatively short.
U.S. Pat. No. 6,203,992 discloses a PCR based method for detecting CK 19 target sequence. Unfortunately, the method disclosed by the '992 patent does not have a desirable ability to accurately quantify the level of CK 19 present in a sample.
There is therefore a need in the art for an improved method and sequences that can be employed according to nucleic acid amplification principles to detect a CK 19 target sequence that provide a greater degree of quantitation.